The ability to deliver genes to the heart and blood vessels, and to manipulate their expression, may make possible the treatment of numerous cardiac disorders. Unfortunately, gene transfer into the heart and blood vessels presents several problems including the relative inaccessibility of the heart tissue, and the fact that cells of the heart are non-dividing, terminally differentiated cells. The standard approach for somatic cell gene transfer, i.e., that of retroviral vectors, is not feasible for the heart, as retrovirally mediated gene transfer requires at least one cell division for integration and expression.
Thus, non-retroviral vectors and non-viral methods, such as the direct injection of "naked" plasmid DNA into the heart, have therefore been used for gene transfer in the heart. The use of "naked" DNA is extremely inefficient for delivering genes to the heart, and expression of the genes transferred by such methods has been largely transient. In addition, naked DNA has only been successfully used to transduce heart cells in vivo after a direct needle injection into the heart muscle during a surgical procedure. This is impractical for widespread cardiac gene therapy and it only influences a small area of cardiac muscle.
A viral vector derived from hemagglutinating virus of Japan (HVJ), which was complexed with liposomes, has been used as an alternative to retroviral vectors and the direct injection of naked DNA (Sawa et al (1994) Circulation 90:I-46, Abstract 0239). However, this system was used in isolated hearts which were to be later transplanted, and was thus only considered to possibly provide new therapy for heart transplantation.
Adenovirus vectors have also been used to transfer genes to the heart, but they have likewise only been applied by direct needle injection into the heart muscle (Duboc et al (1994) Circulation 90:I-517, Abstract 2784; French et al (1994) Circulation 90:I-517, Abstract 2785) or using a rat carotid balloon injury model, in which a solution containing the vector is actually incubated in a blood vessel (Guzman et al (1994) Proc. Natl. Acad. Sci. USA 91:10732-10736). Adenovirus vectors are more efficient at transferring genes into the adult heart than naked DNA, but their expression has likewise only been transient.
Moreover, although adenovirus vectors are designed such that they lack one or more essential viral genes (i.e., the adenovirus E1a immediate early gene) and are thus replication deficient, they retain numerous viral genes which yield expression of viral proteins in addition to the foreign gene of interest. Hence, as a result of the continued production of viral proteins within target cells, significant inflammation can develop. Such inflammation can be an important factor limiting longevity of foreign gene expression. Moreover, this inflammation can be damaging to healthy tissue, which is undesirable if these vectors are intended to protect healthy tissue which is already at risk, or alternatively, if the function of healthy tissue were to be augmented in order to replace the functions lost in nearby damaged tissue. In any event, even in the absence of inflammation, these viral gene products may also be directly toxic to recipient cells. Finally, stability of long-term expression of genes transferred by adenovirus vectors is currently unclear since there is no mechanism for specific viral integration in the genome of non-dividing host cells at high frequency.
Adeno-Associated Virus (AAV) is a defective parvovirus whose genome is encapsidated as a single-stranded DNA molecule. Strands of plus and minus polarity are both packaged, but in separate virus particles. A productive infection requires co-infection by a non-AAV helper virus such as adenovirus or herpes virus, which provides proteins necessary for AAV replication and packaging. In the AAV vector system, 96% of the parental genome has been deleted such that only the terminal repeats remain, containing only recognition signals for DNA replication and packaging. AAV structural proteins are provided in trans by co-transfection with a helper plasmid containing the missing AAV genes but lacking replication/packaging signals.
Although AAV can replicate under special circumstances in the absence of a helper virus, efficient replication requires coinfection with a helper virus of the herpesvirus or adenovirus family. In the absence of the helper virus, AAV establishes a latent infection in which the viral genome exists as an integrated provirus in the host cell. No AAV gene expression is required to establish a latent infection. The integration of the virus is site-specific (chromosome 19). Overall, virus integration appears to have no apparent effect on cell growth or morphology. See Samulski, Curr. Op. Gen. Devel. 3:74-80 (1993). If a latently infected cell line is later superinfected with a suitable helper virus, the AAV provirus is excised and the virus enters the "productive" phase of its life cycle. However, it has been reported that certain AAV-derived transducing vectors are not rescued by adenovirus superinfection.
AAV has been isolated as a nonpathogenic coinfecting agent from fecal, ocular and respiratory specimens during acute adenovirus infections, but not during other illnesses. Although AAV is a human virus, its host range for lytic growth is unusually broad. Latent AAV infections have been identified in both human and nonhuman cells. Cell lines from virtually every mammalian species tested (including a variety of human, simian, canine, bovine and rodent cell lines) can be productively infected with AAV, provided an appropriate helper virus is used (e.g., canine adenovirus in canine cells). Despite this, no disease has been associated with AAV in either human or other animal populations, unlike both HSV and adenovirus.
The genome of AAV-2 is 4,675 bases in length and is flanked by inverted terminal repeat sequences of 145 bases each. These repeats are believed to act as origins for DNA replication. There are two major open reading frames. The left frame encodes at least four non-structural proteins (the Rep group). There are two promoters P5 and P19, which control expression of these proteins. As a result of differential splicing, the P5 promoter directs production of proteins Rep 78 and Rep 68, and the P19 promoter, Rep 52 and Rep 40. The Rep proteins are believed to be involved in viral DNA replication, trans-activation of transcription from the viral promoters, and repression of heterologous enhancers and promoters. The right ORF, controlled by the P40 promoter, encodes the capsid proteins Vp1 (91 kDa), Vp2 (72 kDa) and Vp3 (60 kDa). Vp3 comprises 80% of the virion structure, while Vp1 and Vp2 are minor components. There is a polyadenylation site at map unit 95. For the complete sequence of the AAV-2 genome, see Vastava et al (1983) J. Virol. 45:555-64.
McLaughlin et al (1988) J. Virol. 62:1963-73 prepared two AAV vectors: dl 52-91, which retains the AAV rep genes, and dl 3-94, in which all of the AAV coding sequences have been deleted. It does, however, retain the two 145 base terminal repeats, and an additional 139 bases which contain the AAV polyadenylation signal. Restriction sites were introduced on either side of the signal. A foreign gene, encoding neomycin resistance, was inserted into both vectors. Viral stocks were prepared by complementation with a recombinant AAV genome, which supplied the missing AAV gene products in trans but was itself too large to be packaged. Unfortunately, the virus stocks were contaminated with wild type AAV (10% in the case of dl 3-94) presumably as a result of homologous recombination between the defective and the complementing virus.
Samulski et al (1989) J. Virol. 63:3822-28 developed a method of producing recombinant AAV stocks without detectable wild-type helper AAV. Their AAV vector retained only the terminal 191 bases of the AAV chromosome. In the helper AAV, the terminal 191 bases of the AAV chromosome were replaced with adenovirus terminal sequences. Since sequence homology between the vector and the helper AAV was thus essentially eliminated, no detectable wild-type AAV was generated by homologous recombination. Moreover, the helper DNA itself was not replicated and encapsidated because the AAV termini are required for this process. Thus, in the AAV system, unlike the HSV system, helper virus could be completely eliminated leaving a helper-free AAV vector stock.
Muro-Cacho et al (1992) J. Immunother. 11:231-237 have used AAV-based vectors for gene transfer into both T- and B-lymphocytes. Walsh et al (1992) Proc. Nat. Acad. Sci. (USA) 89:7257-61 used an AAV vector to introduce a human gamma globulin gene into human erythroleukemia cells; the gene was expressed. Flotte et al (1993) J. Biol. Chem. 268:3781-90 delivered the cystic fibrosis transmembrane conductance regulator gene to airway epithelial cells by means of an AAV vector. See also Flotte et al (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-56; Flotte et al (1993) Proc. Nat. Acad. Sci. (USA) 90:10613-17; Kaplitt et al (1994) Nature Genetics 8:148-154).
In view of the aforementioned insufficiencies associated with prior art methods of delivering foreign genes to the heart and blood vessels, it is apparent that there exists a need for such a method which provides safe and stable gene expression, preferably without invasive procedures.